Revision as of 18:06, 17 March 2007

This protocol produces very clean, concentrated DNA, usually on the order of several mg/mL. It is labor intensive. Expect to spend most of the day doing it, but I like to store my plasmids in this form.

Ingredients

Ingredients are per culture; make enough for one extra culture to allow for pipetting error).

Directions

The next morning, put 850μL of the culture in each of two Eppendorf tubes, add 150μL sterile 50% glycerol, and store at -80°C. Pour as much culture as will fit into an Oak Ridge tube and centrifuge at 5800g/6000rpm, 4°C, for 10 minutes in a GSA rotor. Discard the supernatant, add the rest of the culture, and repeat. Resuspend in 1mL TEG.

Add 700μL phenol:chloroform:isoamyl alcohol. Vortex until thoroughly mixed. Centrifuge at top speed of microfuge for 2 minutes. Pipette aqueous phase (the top one) into new Eppendorf. Repeat until the interface between the phases is clear after centrifugation. Then repeat the procedure twice with chloroform:isoamyl alcohol to remove any phenol.